Analytical Methods For 2-Deoxy-D-Glucose

ABSTRACT

2-Deoxy-2-D-glucose (2-DG) concentration and purity can be measured in a sample of crystalline or liquid by HPLC with accuracy and precision suitable for analysis of active pharmaceutical ingredient and drug product.

BACKGROUND OF THE INVENTION

1. Field of Invention

The present invention provides methods for analysis of purity andconcentration of 2-deoxy-D-glucose (2-DG), especially in preparationsintended for therapeutic use, and so relates to the fields of chemistry,biology, pharmacology, and medicine.

2. Description of Related Art

2-Deoxy-D-glucose (2-DG) has been studied to determine if the compoundhas potential application as an anticancer agent (see Blough et al.,1979, JAMA 241 (26): 2798, incorporated herein by reference). Recentadvances, as described in PCT patent application No. US04/000530 andU.S. Pat. No. 6,670,330, both of which are incorporated herein byreference, that are being implemented in ongoing clinical trialsindicate that 2-DG should prove to be a useful anticancer agent.Employing 2-DG as an active pharmaceutical ingredient (API) in a drugproduct requires an accurate method for determining the concentrationand purity of 2-DG.

HPLC analysis has been used to determine the concentration and purity ofglucoase, a 2-DG analog. Columns and chromatographic conditions thathave been described for the analysis of glucose using a refractive index(RI) detector are shown in Table 1, below.

TABLE 1 Mobile Vendor Column Temperature Phase Flow rate Alltech AstecAmino Ambient ACN:water 1 mL/min 250-4.6 mm (75:25) 5-μm AlltechHypersil Ambient ACN:water 0.5 mL/min APS-2 (80:20) 100 × 3.2 mm 5-μmPhenomenex Luna Amino 40° C. ACN:water 3 mL/min 250 × 4.6 mm (80:20)5-μm Phenomenex Rezex 85° C. Water 0.6 mL/min RCM-Mono- saccharide 300 ×7.8 mm

One of the methods used for determining the purity of 2-DG in a sampleis gas chromatography (GC; see Blough et al., supra, page 2799).However, 2-DG is a non volatile, high melting solid and needs to betransformed chemically into a volatile derivative that can be evaporatedfor analysis by GC. The transformation procedure involves reacting 2-DGwith a trimethylsilylating agent, and the purity of its volatiletrimethylsilylated derivative is actually analyzed by GC. The purity of2-DG in the sample is thus indirectly inferred from the analysis of thederivative. In one approach, 2-DG has been reacted withtrimethylsilylimidazole and pyridine for five minutes in an all glassreaction-vessel, prior to GC analysis (Blough et al., supra).

The drawbacks to this method include the following. Because there arefour hydroxy groups in 2-DG that can be trimethylsilylated, each of themhas to react with trimethylsilyl chloride (or any othertrimethylsilylating agent), thus yielding a single product (which isanalyzed in comparison to other components in the chromatogram), todescribe the purity of 2-DG accurately. If the silylation reaction isincomplete, the formation of partially silylated derivatives canerroneously diminish the measured purity or concentration of the 2-DG inthe sample. Also, the silylation product has to be stable during theprocess of evaporation and passage through the column at hightemperatures, and the reactive 1′-TMS ether may become deprotectedduring this process.

In another method, 2-DG in rat serum has been analyzed by HPLC followinga post column fluorescence derivatization (see Umegae et al., 1990,Chem. Pharm. Bull. 38 (4): 963-5, incorporated herein by reference). Inthis method, the sugars are converted into fluorescent derivatives byreaction with meso-1,2-bis(4-methoxyphenyl)ethylenediamine in analkaline medium after separation on a strong anion exchange column (TSKgel Sugar AXG), and the fluorescent analogs are analyzed by afluorescent detector. The detection limit in one application was, at asignal-to-noise ratio of 3, 0.52 nmol/mL. Again, the requirement of areactive step and the measurement of an entity different from the actualanalyte are among the drawbacks of this method.

Another method for analyzing the presence of tritiated ³H-2-DG in ratmuscle using chromatography has been reported (see Wallis et al., 2002,Diabetes, 51:3492, incorporated herein by reference). In this method,free and phosphorylated ³H-2-DG are separated by ion exchangechromatography using an anion exchange resin (AG1-X8). Biodegradablecounting scintillant, BCA (Amersham), is added to each radioactivesample and radioactivity determined using a scintillation counter(LS3801; Beckman). However, the radioactivity of 2-DG is used as aread-out, so the method is useful only for radio-labeled 2-DG.

Another method for determining 2-DG purity, in topical formulations,that involves HPLC has been employed with ultraviolet detection (UV) at195 nm (see Hughes et al., 1985, J. Chromatogr. 331(1):183-6,incorporated herein by reference). 2-DG does not possess a chromophoreabsorbing above 200 nm, and a very low wave-length of 195 was chosen bythe scientists reporting the method for the purpose of analysis. Columnsthat have been used in the method are a μBondapak 10 μm NH₂ column and aVarian Micropak 10 μm NH₂ column. The eluent used was 85% MeCN/H₂O. Theretention time of 2-DG reported in one application was about 4 minutes.Such a retention time is typically too short to observe impuritiespresent in the sample, especially if the impurities are structurallyclosely related compounds like glucose.

There remains a need for methods for analyzing the purity andconcentration of 2-DG that do not require derivatization, provideaccurate results, especially at low concentrations, and are applicableto crystalline 2-DG. The present invention meets these needs.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a method of separating 2-DG employinganion exchange chromatography wherein the anion exchange chromatographyuses a poly(styrene-divinylbenzene) based polymer as a stationary phase.In one embodiment, the poly(styrene-divinylbenzene) based stationaryphase contains ammonium groups. In a related embodiment, the ammoniumgroup is a trimethylammonium group. In one embodiment, apoly(methylacrylamido propyl trimethylammonium salt) based polymerprovides the trimethylammonium employed in the stationary phase.Examples for separating 2-DG employing anion exchange chromatographywherein the anion exchange chromatography usespoly(styrene-divinylbenzene) based stationary phases includes anionexchange chromatography employing RCX-10, RCX-30, and Aminex HPX-87Xanion exchange columns. Examples of separating 2-DG employing anionexchange chromatography wherein the anion exchange chromatography usespoly(styrene-divinylbenzene) based stationary phases containingtrimethylammonium groups include anion exchange chromatography employingRCX-10 and RCX-30 anion exchange columns.

In one aspect, the present invention provides an HPLC-based method foranalyzing the purity of crystalline 2-DG, said method comprising thesteps of: (a) dissolving said crystalline 2-DG in an aqueous solution;(b) chromatographing a sample of said aqueous 2-DG solution on an ionexchange column using an eluent selected from the group consisting ofwater, aqueous alkali, and aqueous acid as; (c) measuring an amount of2-DG and any impurities in said sample after said chromatography bymeans of a detector that generates a signal proportional to the amountof said 2-DG in said sample; and (d) determining the purity of saidcrystalline 2-DG by comparing the signal generated by said 2-DG with anysignal generated by said impurities in said sample.

In one embodiment, an anion exchange column and aqueous alkali eluentare employed. In another embodiment, an ion exchange column and aqueousacid eluent are employed. In another embodiment, an ion exchange columnand water eluent are employed. In another embodiment, an anion exchangecolumn and aqueous alkali eluent are employed, and an RI detector or apulsed amperometric detector (PAD) is used to generate the signal. Inone embodiment, an RI detector or a pulsed amperometric detector is usedto generate the signal, and the crystalline 2-DG solution analyzedcontains between about 1 μg/mL and 10 mg/mL of crystalline 2-DG.

In another aspect, the present invention provides an HPLC method foranalyzing the purity of 2-DG in an aqueous solution, said methodcomprising the steps of: (a) chromatographing a sample of said aqueous2-DG solution on an ion exchange column using an eluent selected fromthe group consisting of water, aqueous alkali, and aqueous acid; (b)measuring an amount of 2-DG and any impurities in said sample after saidchromatography by means of a detector that generates a signalproportional to the amount of said 2-DG in said sample; (c) determiningthe purity of said 2-DG by comparing the signal generated by said 2-DGwith any signal generated by said impurities in said sample. In oneembodiment, the detector is a detector other than a UV detector.

In one embodiment, an anion exchange column and aqueous alkali eluentare employed. In another embodiment, an ion exchange column and aqueousacid eluent are employed. In another embodiment, an ion exchange columnand water eluent are employed. In another embodiment, an anion exchangecolumn and aqueous alkali eluent are employed, and an RI detector or apulsed amperometric detector PAD is used to generate the signal. Inanother embodiment, an RI detector or a pulsed amperometric detector isused to generate the signal, and said 2-DG solution contains betweenabout 1 μg/mL and 10 mg/mL of 2-DG.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a chromatogram for 2-DG (2 mg/mL) and glucose (2 mg/mL).

FIGS. 2A and 2B show chromatograms for blank injections of water (FIG.2A) and mobile phase (see FIG. 2B).

FIGS. 3A and 3B show chromatograms. FIG. 3A is a chromotogram for aplacebo (1.8 mg/ml methylparaben and 0.2 mg/ml propylparaben); FIG. 3Bis a chromatogram for the same sample after degradation by exposure to70° C. for 1 day.

FIG. 4 shows a chromatogram for 2-deoxyglucose (2-DG) after 35 days at60° C.

FIG. 5 shows a chromatogram for 2-DG after 23 days at 60° C.

FIGS. 6A and 6B show chromatograms for 2-DG after degradation byincubation for 5 days at 60° C. at pH 2, and pH 5, respectively.

FIGS. 7A and 7B show chromatograms for oxidized 2-DG samples. The samplein FIG. 7A is 5 ml 2-DG+50 μl H₂O2 after storage at 60° C. for 17 hours.The sample in FIG. 7B is 5 ml 2-DG+100 μl H₂O2 after storage at 60° C.for 17 hours.

FIGS. 8A and 8B are chromatograms for 20 mg/ml 2-DG samples, after beingdegraded by exposure to intense fluorescent light for 35 days.

FIG. 9 shows average peak area for 1 to 3 mg/ml samples of 2-DG inwater.

FIGS. 10A and 10B show average peak area for 0.1-1.2 mg/ml glucose inassays run with 10 μl samples (FIG. 10A) and for 0.01-0.12 glucose inassays using 80 μl samples (FIG. 10B).

FIG. 11 shows a chromatogram for 10 μg/ml glucose.

DETAILED DESCRIPTION OF THE INVENTION Example 1 Assay of 2-DG andRelated Compounds in API and Drug-Product

This example illustrates how 2-DG purity was assessed in a mixturecontaining 2-DG and glucose in accordance with an embodiment of themethod of the invention in which aqueous NaOH was the mobile phase, ananion exchange column was the stationary phase, an RI detector wasemployed, and the concentration of 2-DG in the 2-DG solution analyzedwas about 2 mg/mL. A sample of 2-DG drug product was prepared bydissolving API grade 2-DG into an aqueous solution containingmethylparaben (0.18%) and propylparaben (0.02%). Chromatographicparameters analyzed to illustrate the method included system linearity,accuracy, system precision, system suitability, limits of detection andquantitation, and robustness and ruggedness.

The general procedure for HPLC employed an isocratic HPLC method, withan RI detector equipped with an anion-exchange column (Hamilton RCX-10,250×4.1 mm, 0 7-μm) controlled at 30° C. The mobile phase was 18 mM NaOHin water and a flow rate of 0.7 mL/min yielded baseline resolution of2-DG and glucose.

The method was performed using a Shimadzu HPLC system equipped with anautomatic data acquisition system (ChromPerfect), a Shimadzu pump (ModelLC-10AD), a Shimadzu autosampler (Model SIL-10A) and an RI detector(Agilent model 1100). The materials employed in the analyses, along withtheir suppliers are listed below:

Sodium hydroxide ACS Grade 2-deoxy-D-glucose Ferro-Pfanstiehl2-deoxy-D-glucose Ferro-Pfanstiehl 2-deoxy-D-glucose* Sigma Glucose*Sigma Methylparaben Sigma Propylparaben Sigma Water Milli-Q water *Thereference standard employed in the experiment.

Determination of Specificity

The placebo solutions and the solutions used for specificity andstability measurements were prepared as follows. The placebo solutionwas prepared by warming an appropriate mixture of methylparaben andpropylparaben in water to about 70° C. and diluting this solutionquantitatively. A solution of API 2-DG was prepared by dissolvingcrystalline 2-DG in water. A solution of 2-DG drug-product was preparedby dissolving a sample of crystalline 2-DG in the placebo solution.

A typical chromatogram for 2-DG and glucose, each at 2 mg/mL, is shownin FIG. 1. Under the conditions of the method, 2-DG eluted at about 8minutes, and glucose eluted between 9 and 10 minutes. Peaks elutingbefore 6 min were system peaks, which showed some variabilityrun-to-run. Resolution between 2-DG and glucose was 2.4 with 3100theoretical plates for both peaks. Both 2-DG and glucose peaks werewell-shaped with an asymmetry (tailing) of 1.7.

The methods of the invention can be useful in measuring the heatstability of an aqueous API 2-DG solution. In one test, heat stabilitywas determined by storing the solution at 60° C. for 35 days in a sealed2 mL glass vial. The methods of the invention can also be useful inmeasuring the light stability of an aqueous API 2-DG solution. In onetest, light stability was determined by exposing the solution to intensefluorescent light for 35 days in a sealed 2 mL glass vial.

The chromatograms for blank injections of water (see FIG. 2A) and mobilephase (see FIG. 2B), placebo containing methylparaben at 1.8 mg/mL andpropylparaben at 0.2 mg/mL (see FIG. 3A), and placebo degraded at 70° C.for one day (see FIG. 3B) demonstrated that the background signal didnot interfere with the quantitation of 2-DG or glucose peaks. API ordrug-product 2-DG was exposed to elevated heat (see FIGS. 4 and 5respectively), acid/base (see FIGS. 6A and 6B), oxidation by H₂O₂ (seeFIGS. 7A and 7B) and intense fluorescence light (see FIGS. 8A and 8B).The results showed there was no degradation in samples exposed to 60° C.or intense fluorescent light for at least 35 days; that 2-DG was stablein pH 2 or pH 10 solution stored at 60° C. for 5 days; and that therewas approximately 23% and 34% degradation in 50 and 100 μL H₂O₂ added2-DG solutions stored at 60° C. for 17 days.

System Linearity

To determine system linearity for 2-DG, a series of 2-DG standardsolutions in water, in the concentration range of 50-150% of theexpected injectate concentration (2 mg/mL), were prepared. Triplicateinjections were made for each solution. Six replicate injections weremade for the injected concentration at about 2 mg/mL. Excellentlinearity was observed for the measured peak area versus 2-DGconcentration in the injectate, with an r² value of 0.9999, a slope of231797 and a y-intercept of 8179 (see Table 2 and FIG. 9).

The system linearity for glucose was performed by preparing a series ofglucose standard solutions in water in the concentration range of0.1-1.2 mg/mL with 10 μL injection (see Table 3A and FIG. 10A) and10-120 μg/mL with 80 μL injection (see Table 3B and FIG. 10B). Excellentlinearity was observed for the measured peak area versus glucoseconcentration in the injectate, with r² values of 0.9998 and 0.9997,respectively.

TABLE 2 System Linearity of 2-DG 2-DG Concentration % of Nominal (mg/mL)(2 mg/mL) Peak Area Mean ± SD 1.001 50.1% 240406 241927 ± 5011 247522237852 1.603 80.2% 376265 376437 ± 4117 372409 380638 1.982 99.1% 468109468228 ± 2531 467918 468014 467427 465429 472352 2.412 120.6% 565828568212 ± 2116 568940 569868 3.030 151.5% 707758 710550 ± 4102 715259708633 Slope = 231797 Y-intercept = 8179 R² = 0.9999

TABLE 3A System Linearity for Glucose (10 μL Injection) GlucoseConcentration % of Nominal (mg/mL) (2 mg/mL) Peak Area Mean 0.1  5%22483 24661 26838 0.4 20% 93967 94815 95662 0.8 40% 188393 187668 1869431.2 60% 281154 286404 291653 Slope = 238348 Y-intercept = −104 R² =0.9998

TABLE 3B System Linearity for Glucose (80 μL Injection) GlucoseConcentration % of Nominal (μg/mL) (2 mg/mL) Peak Area Mean 10.21 0.51%19163 19163 21.35 1.07% 42408 40877 39345 40.07 2.00% 78327 80533 8273883.00 4.15% 160186 160622 161057 119.5 5.98% 231600 229933 228265 Slope= 1925 Y-intercept = 710 R² = 0.9997

Determination of System Precision

A 2-DG standard solution at 1.98 mg/mL was injected six times and thepeak areas (mAU•sec) determined (see Table 4). The relative standarddeviation (RSD) was 0.5%.

TABLE 4 System Precision Peak Sample No. Area(mAU · sec) Mean ± SD RSD 1468109 2 467918 3 468014 468228 ± 2531 0.5% 4 467427 5 465429 6 472352

Determination of System Suitability

System suitability was determined by six replicate injections of asystem suitability-resolution solution. The RSD of the peak area andretention time of 2-DG were 0.8% and 0.0%, respectively. The RSD of thepeak area and the retention time of glucose were 0.7% and 0.0%,respectively (see Table 5). The average resolution between 2-DG andglucose was 2.79±0.01 (n=6).

TABLE 5 System Suitability of 2-DG and Glucose Glucose 2-DG 2-DG GlucoseRetention Injection Peak Area Retention Peak Area Time Reso- No. (mAU ·S) Time (min) (mAU · S) (min) lution 1 458700 8.8 489136 10.6 2.78 2453843 8.8 493462 10.6 2.78 3 458488 8.8 491759 10.6 2.80 4 454905 8.8489158 10.6 2.79 5 458445 8.8 492504 10.6 2.80 6 451052 8.8 484803 10.62.79 Mean 453347 8.8 490337 10.6 2.79 SD 3821 0.0 3482 0.0 0.01 RSD 0.8%0.0% 0.7% 0.0% 0.4%

Determination of Accuracy

A known amount of 2-DG reference standard was dissolved in placebo toyield solutions containing 2-DG at 80, 100, and 120 mg/mL. Triplicatesamples were prepared for each concentration. Solutions were diluted to2 mg/mL with water and assayed. The accuracy of this method wasdetermined by evaluating solutions of 2-DG at concentrations of 80%, 100% and 120% of solutions at 100 mg/mL. Recoveries were in the range of101.3-102.8% (see Table 6).

TABLE 6 Accuracy (Nominal Concentration: 100 mg/mL) % of 2-DGConcentration mg/mL % Nominal Expected Found Recovery Mean ± SD  80%77.65 81.86 102.8 102.4 ± 0.8 79.07 80.24 101.5 79.47 81.72 102.8 100%99.02 100.90 101.9 102.2 ± 0.5 98.40 101.15 102.8 99.18 101.14 102.0120% 118.8 120.98 101.8 101.5 ± 0.3 118.1 119.76 101.4 119.5 121.02101.3

Determination of Method Precision

Method precision was assessed by assaying two API lots on four differentdays in the same laboratory. The same HPLC system and column were usedfor all assays. The results indicate that the percent purity in bothlots was very similar on four assay days, and that the method had goodprecision (see Table 7).

TABLE 7 Method Precision (2-DG API) % Purity Assay Date Lot 28445A Lot28506A Mar. 6, 2003 98.0 98.9 Mar. 7, 2003 97.6 98.7 Mar. 13, 2003 97.999.4 Mar. 21, 2003 98.6 99.2 Mean = 98.0 99.1 SD = 0.4 0.3

Limit of Detection and Quantitation of Glucose

A signal-to-noise (S/N) ratio of 3:1 is generally defined as the limitof detection. The S/N ratio for an 80-μL injection of glucose sample at10 μg/mL (or 0.5% of 2-DG at 2 mg/mL), was determined to be 6.7 (FIG.11). Therefore the limit of detection (LOD, defined as 3•S/N) wascalculated to be:

-   10 μg/mL×(3/6.7)=4.5 μg/mL. The limit of quantitation (LOQ, defined    as 10•S/N) was 15 μg/mL.

Ruggedness and Robustness

The 2-DG standard and resolution solutions at a nominal concentration of2 mg/mL were re-assayed versus a freshly-prepared standard solution. Theresults showed both solutions were stable after storage at ambient roomtemperature for 4 days (see Table 8A. 2-DG injectate solutions from twolots were re-assayed after stored at 5° C. for 7 days. The resultsindicate both solutions were stable (see Table 8B.

TABLE 8A Robustness/Ruggedness: Stability of Standard and ResolutionSolutions 2-DG Concentration (mg/mL and % of Initial) Initial 4 days RTStandard Solution 2.026 mg/mL 2.035 mg/mL (≈2 mg/mL) (100.0%) (100.4%) Resolution Solution 2.158 mg/mL 2.125 mg/mL (≈2 mg/ml) (100.0%) (98.5%)

TABLE 8B Robustness/Ruggedness: Stability of Injectate Solutions 2-DGConcentration (mg/mL and % of Initial) Initial 7 days at 5° C. Lot #28445A 2.07 mg/mL 2.03 mg/mL (100.0%) (98.1%) Lot # 28506A 2.02 mg/mL1.98 mg/mL (100.0%) (98.0%)

The effects of variation of the NaOH concentration in the mobile phase,column temperature (25° C. and 35° C.), and flow rate (0.6, 0.8 and 1.0mL/min), on 2-DG retention time, and the resolution between 2-DG andglucose (see Tables 9A and 9B were also determined. Variation in 2-DGretention time was observed with chromatography conditions, but in allcases, the resolution was greater than 2.0.

TABLE 9A Robustness/Ruggedness: Effects of Variation on the NaOHConcentration in Mobile Phase, Column Temperature and Flow Rate on 2-DGRetention Time 2-DG Retention Time Mobile Column (min) with Flow Rate atPhase Temperature 0.6 mL/min 0.8 mL/min 1.0 mL/min 20 mM NaOH 35° C.9.09 7.35 6.17 16 mM NaOH 25° C. 11.24 8.32 6.54

TABLE 9B Robustness/Ruggedness: Effects of Variation on the NaOHConcentration in Mobile Phase, Column Temperature and Flow Rate onResolution of 2-DG and Glucose Mobile Column Resolution with Flow Rateat Phase Temperature 0.6 mL/min 0.8 mL/min 1.0 mL/min 20 mM NaOH 35° C.2.55 2.60 2.54 16 mM NaOH 25° C. 2.81 2.61 2.43

Example 2

This example illustrates how 2-DG purity was assessed in a mixturecontaining 2-DG, glucose, and tri-O-acetyl-D-glucal (glucal), inaccordance with an embodiment of the method of the invention in whichaqueous NaOH was the mobile phase, an RCX-10 anion exchange column wasthe stationary phase, an electrochemical (EC) detector was employed, andthe concentration of 2-DG in the 2-DG solution analyzed was about 10μg/mL. Acceptable separation of 2-DG and glucose was obtained with 10-50mM NaOH being employed as the mobile phase. An increase in NaOHconcentration decreased retention time for 2-DG and glucose. With 47 mMNaOH in the mobile phase, the following result was obtained (see Table10).

TABLE 10 2-DG glucose glucal Concentration 10 μg/mL 1 μg/mL 50 μg/mLPeak Area 17,683,388 15,033,551 Retention Time 8.6 min 10.2 min 14.8 minNote Good sharp peak Good sharp peak Slight tailing

Example 3

This example illustrates how 2-DG purity was assessed in a solutioncontaining 2-DG, glucose, and glucal in accordance with an embodiment ofthe method of the invention in which aqueous NaOH was the mobile phase,an RCX-30 anion exchange column was the stationary phase and an ECdetector was employed (see Table 11). The peak corresponding to glucaldissolved in 30 mM NaOH (50 μg/mL) was a sharp large peak with retentiontime at about 11 minutes, possibly because of a hydrolysis of the glucalto 2-DG in the alkaline solution. However, the same sample dissolved inwater resulted in a poorly-shaped, small peak.

TABLE 11 Mobile Phase Retention Time Retention Time (NaOH) SampleDissolved in (2-DG) (glucose) 40 mM water 10 min 14 min 30 mM water 13min 18 min 40 mM 30 mM NaOH 9-10 min 13 min

Example 4

This example illustrates how 2-DG purity was assessed in a mixturecontaining 2-DG and glucose in accordance with an embodiment of themethod of the invention in which aqueous acid was the mobile phase, anaminex column was the ion exchange column and an EC detector wasemployed (see Table 12). This example further illustrates how 2-DGpurity was assessed in a solution containing 2-DG and glucal inaccordance with an embodiment of the method of the invention in whichwater was the mobile phase, an aminex column was the ion exchangecolumn, and an EC detector was employed.

TABLE 12 Column Mobile Phase Retention time Aminex 0.009N H₂SO₄  8.4 min(glucose) HPX-87H  9.5 min (2-DG) Aminex water 13.3 min (glucal) HPX-87N10.2 min (2-DG)

1. An HPLC method for analyzing purity of crystalline 2-deoxy-D-glucose(2-DG), said method comprising the steps of: (a) dissolving saidcrystalline 2-DG in an aqueous solution; (b) chromatographing a sampleof said aqueous 2-DG solution on an ion exchange column using an eluentselected from the group consisting of water, aqueous alkali, and aqueousacid; (c) measuring an amount of 2-DG and any impurities in said sampleafter said chromatography by means of a detector that generates a signalproportional to the amount of said 2-DG in said sample; and (d)determining the purity of said crystalline 2-DG by comparing the signalgenerated by said 2-DG with any signal generated by said impurities insaid sample.
 2. The method of claim 1, wherein said chromatographyperformed in step (b) employs an anion exchange column and aqueousalkali eluent.
 3. The method of claim 1, wherein said chromatographyperformed in step (b) employs an ion exchange column and aqueous acideluent.
 4. The method of claim 1, wherein said chromatography performedin step (b) employs an ion exchange column and water eluent.
 5. Themethod of claim 1, wherein said chromatography performed in step (b)employs an ion exchange column and water eluent.
 6. The method of claim5, wherein said aqueous solution contains between 1 μg/mL and 10 mg/mLof said crystalline 2-DG.
 7. An HPLC method for analyzing purity of 2-DGin an aqueous solution said method comprising the steps of: (a)chromatographing a sample of said aqueous 2-DG solution on an ionexchange column using an eluent selected from the group consisting ofwater, aqueous alkali, and aqueous acid; (b) measuring an amount of 2-DGand any impurities in said sample after said chromatography by means ofa detector that generates a signal proportional to the amount of said2-DG in said sample but that is not an ultra-violet detector; and (c)determining the purity of said 2-DG by comparing the signal generated bysaid 2-DG with any signal generated by said impurities in said sample.8. The method of claim 7, wherein said chromatography performed in step(b) employs an anion exchange column and aqueous alkali eluent.
 9. Themethod of claim 7, wherein said chromatography performed in step (b)employs an ion exchange column and aqueous acid eluent.
 10. The methodof claim 7, wherein said chromatography performed in step (b) employs anion exchange column and water eluent.
 11. The method of claim 8, whereinsaid detector of step (c) is an RI detector or a pulsed amperometricdetector.
 12. The method of claim 11, wherein said aqueous solutioncontains between 1 μg/mL and 10 mg/mL of 2-DG.
 13. The method of claim1, wherein said crystalline 2-DG is a sample of active pharmaceuticalingredient.
 14. The method of claim 7, wherein a concentration of 2-DGin said sample is determined.
 15. The method of claim 7, wherein saidsample of 2-DG is a sample of a drug product.